Insights into the structure and nanomechanics of the Quatsome membrane by force spectroscopy measurements and molecular simulations

Quatsomes (QSs) are unilamellar nanovesicles constituted by quaternary ammonium surfactants and sterols in defined molar ratios. Unlike conventional liposomes, QS are stable upon long storage such as for several years, they show outstanding vesicle to vesicle homogeneity regarding size and lamellarity, and they have the structural and physicochemical requirements to be a potential platform for site specific delivery of hydrophilic and lipophilic molecules. Knowing in detail the structure and mechanical properties of the QS membrane is of great importance for the design of deformable and flexible nanovesicles alternatives, highly pursued in nanomedicine applications like the transdermal administration route. In this work, we report the first study on the detailed structure of the Cholesterol:CTAB QS membrane at the nanoscale, using atomic force microscopy (AFM) and spectroscopy (AFM-FS) under controlled liquid environment (ionic medium and temperature) to assess the topography of supported QS membranes (SQMs) and to evaluate the local membrane mechanics. We further perform molecular dynamics (MD) simulations to provide an atomistic interpretation of the obtained results. Our results are direct evidence of the bilayer nature of the QS membrane, with characteristics of a fluid-like membrane, compact and homogeneous in composition, and which structural and mechanical properties depend on the surrounding environment. We show how ions alter the lateral packing, modifying the membrane mechanics. We observe that according to the ionic environment and temperature, different domains may coexist in the QS membranes, ascribed to variations in molecular tilt angles. Our results indicate that QS membrane properties

may be easily tuned by altering the lateral interactions either with different environmental ions or counterions.

INTRODUCTION
The field of nanomedicine has grown rapidly in the recent years.Great efforts have been addressed towards new drug delivery systems that may improve the administration of drugs in a more effective and safe manner, increasing their solubility and stability, overcoming the anatomical and physiological barriers by targeting specific organs/tissues, reducing their quick clearance from the body and therefore, their side effects. 1,2 4][5][6] Liposomes are molecular self-assembled lipid-based nanovesicles, undoubtedly one of the most promising supramolecular assemblies for nanomedicine due to their great versatility with respect to size, composition, surface characteristics and capacity for integrating and encapsulating bioactive molecules, both hydrophobic and/or hydrophilic.Their membranes can be efficiently functionalized with ligands with different hydrophobicity, and different targeting units like peptides, antibodies, etc.Moreover, liposomes are well recognized as pharmaceutical carriers because of their biocompatibility, biodegradability and low toxicity. 7,8  physicochemical and mechanical properties of the nanoparticles are key as they may affect the interaction with cells and tissues, the endocytic pathway, 9 the drug permeability, and the deformability, which is essential for some applications, like skin penetration. 10In lipid nanovesicles, the deformability and mechanical properties of the vesicles is directly related to the membrane structure and rigidity. 11,12 24][25][26] Still, one of the major problems limiting the widespread use of liposomes is their poor stability, both physical (colloidal), including aggregation or fusion of vesicles to form larger and heterogeneous particles, and chemical, i.e. hydrolysis of ester groups and oxidation of unsaturated chains. 27Liposomes with higher stability generally comprise gel phospholipids, and therefore deformability is compromised.The need for alternative vesicular systems with enhanced properties compared to liposomes, especially when vesicles' mechanical properties are a critical issue, has led to the design of alternative lipid nanovesicles, like the Quatsomes (QS).
QS are unilamellar nanovesicles constituted by quaternary ammonium surfactants and sterols in defined molar ratios. 8,28,29 Ulike conventional liposomes, QS are stable upon long storage such as for several years, and they show outstanding vesicle to vesicle homogeneity regarding size and lamellarity. 29,30 [33][34] QS-like structures have been prepared using different quaternary ammonium surfactants such as cetrimonium bromide (CTAB), myristalkonium chloride (MKC) and cetylpyridinium chloride (CPC) and different sterols such as cholesterol (Chol) and b-sitosterol. 28,29 ng molecular dynamic (MD) simulations, it has been demonstrated that, in an aqueous environment, the synergy between the cetyltrymethyl ammonium (CTA + ) of CTAB and Chol molecules makes them self-assemble into bimolecular amphiphiles (synthon) and then into bilayers, with a similar structure of those formed by double-tailed unimolecular amphiphiles, like phospholipids. 29However, a detailed characterization on the structure and mechanical properties of the QS membrane has not been performed.Local characterization of these membrane properties can be widely explored with techniques that allow working at nanometric resolution and preserving the physiological membrane environment.In this context, atomic force microscopy (AFM), 35 AFM-based force spectroscopy (AFM-FS) 14,36 and force clamp (AFM-FC) 37,38 are essential tools to locally study the physical properties of supported membranes at the nanoscale with high spatial range sensitivity and versatility while giving the possibility to control the environmental conditions.Lateral interactions between the molecules can be directly evaluated with AFM-FS, by measuring the maximum vertical force a membrane is able to resist before its rupture, the breakthrough force F b , when indented by the AFM tip.26 Here, we study for the first time the detailed structure of the QS membrane at the nanoscale, using AFM and AFM-FS under controlled liquid environment (ionic medium and temperature) to assess the topography of supported QS membranes (SQMs) and to evaluate the local membrane mechanics.We further perform MD simulations to provide an atomistic interpretation of the obtained results.Our results are direct evidence of the bilayer nature of the QS membrane, with characteristics of a fluid-like membrane, and which structural and mechanical properties depend on the surrounding environment.We demonstrate how ions alter the lateral packing, modifying the membrane mechanics.According to the ionic environment and temperature, we show that different domains may coexist in the QS membranes, ascribed to variations in molecular tilt angles.

Supported QS membrane structural characterization
For the study at the nanoscale of Chol:CTAB QS bilayer and its mechanical properties, we prepared two different supported QS membranes (SQMs) on mica surfaces using the QS vesicular samples described in Table 1.The samples were prepared following the DELOS-susp procedure, 31 a one-step methodology based on the use of compressed CO 2 which permits the straightforward preparation of the nanoscopic QSs, without further steps of extrusion or thawfreezing.The mean size of QSs obtained were in the range between 90-150 nm in diameter.The vesicles structural characterization by dynamic light scattering (DLS) and Cryo-TEM are detailed in the SI (Table S1, Figure S1).forming SQMs.This procedure is equivalent to the liposome rupture method 43 based on the formation of a supported lipid bilayer (SLB) by depositing a suspension of liposomes onto a flat surface.This allows for a detailed morphological and nanomechanical characterization at the nanoscale, using atomic force microscopy (AFM) and spectroscopy (AFM-FS), under controlled liquid environment.

Topographical characterization
As shown in Figure 1, QS_H 2 O and QS_PBS membranes spread all over the mica surface.At the initiation of the AFM experiment (t 0 ), after depositing the QSs onto the mica surface for 30 min at room temperature (RT), coexistence of domains of different thickness was identified for SQMs in both liquid environments.The thickness of the different domains was determined from the force-separation curves (Figure S2) performed during the AFM-FS measurements: 4.7 ± 0.2 nm and 4.1 ± 0.3 nm for the higher and lower domains for QS_H 2 O membranes, and 5.3 ± 0.4 nm and 4.5 ± 0.4 nm for the higher and lower domains for QS_PBS membranes.SQM in PBS/NaCl are slightly thicker than in water.Conversely, the QS_PBS membrane showed a dynamic behavior towards a homogeneous bilayer after 30 min (t f ) (Figure 1, bottom).This effect was not a consequence of the imaging as it was verified when imaging different unexplored areas of the same sample after t f .The membrane at this state had a thickness of 4.9 ± 0.5 nm.

Supported QS membrane nanomechanics
Lateral interactions between the molecules of 2D ordered structures like lipid bilayers can be explored by measuring the maximum force the membrane is able to withstand before its rupture, because of an applied external pressure.In an AFM-FS experiment, the AFM tip breaks through the membrane at a force (the breakthrough force F b , see Figure S2) 14,15,36,44 that is characteristic of the lateral packing of the membrane of certain composition in a particular environment and at a specific tip velocity.
Force-separation curves were performed over an area of the SQM previously imaged, and we clearly observed the breakthrough events as sharp discontinuities on the approach forceseparation curves (Figure S2 and Figure 2c).These sharp breakthroughs at a few nNs are generally characteristic of fluid-like lipid bilayers. 42,45,46 W calculated the F b values at each pixel and built the F b maps directly correlating the AFM topography.The F b value of each sample was determined by fitting the F b distributions to a Gaussian model (Figure 2d).The reported mean F b for each membrane corresponds to the average of F b values of 10 samples ± SD (Figure 3).
For QS_H 2 O, a bimodal F b distribution (Figure 2d) is obtained, with mean values of 1.2 ± 0.5 nN and 1.9 ± 0.9 nN.Although the difference is not large, each value is associated to each of the domains, as evidenced in the correlation of the F b map (Figure 2b) and the topography (Figure 2a), with slightly higher F b for the thicker domain than for the thinner one.In PBS/NaCl, the initial heterogeneous topography of QS_PBS SQM (Figure 2a, t 0 ) was also revealed in the F b map (Figure 2b) with a bimodal F b distribution, with mean values of 2.5 ± 0.8 nN and 6.3 ± 2.5 nN for the thinner and thicker domains, respectively (Figure 2b-d).Accordingly, at t f , when the QS_PBS membrane became homogeneous (Figure 2a, t f ), a uniform F b map (Figure 2b-d) with mean value of 6.2 ± 1.8 nN was obtained, comparable to the value of the thicker domain at t 0 .
From these data, it becomes clear that the ions in solution play a key role in the lateral packing of the CTA + and Chol molecules, leading to an increase in the mechanical resistance of the SQM.

Role of the ions on the QS membrane topography and nanomechanics
From the topographical and nanomechanical characterization it is evident that the ions present in the liquid environment when the QS assembly takes place are affecting the membrane structure.
Not only they may adsorb on the QS surface, but also affect the lateral packing, as the membrane in ionic media shows a higher resistance to break upon indentation.[46][47][48][49] To better understand the effect of ions into SQMs, we assessed the topographical and mechanical properties of the QS_H 2 O membrane before and after replacing the liquid environment from water to PBS/NaCl.Figure 4 shows the topography, F b maps and histograms for these two cases.
From the QS_H 2 O membrane with coexisting domains and bimodal F b distribution, after rinsing several times with PBS/NaCl, the mechanical stability increases to F b values close to those of QS_PBS membranes (3.4 ± 0.6 nN for QS_H 2 O + PBS, Figure 4).In addition, after PBS/NaCl has been added, the membrane topography and mechanics evolve in time (after ca.50 min) to the one of the SQM from QS_PBS (Figure S4).This means that the ions of the buffer not only affect the membrane properties when QS are produced in PBS/NaCl medium.Ions can also enter and alter the membrane structure upon exposure to the ionic environment, providing a direct evidence that the ions are the direct responsible for the enhancement in lateral interaction and the changes in the membrane mechanics.This atomistic interpretation is confirmed by all-atomic MD simulations in section 2.4.

Effect of the temperature on the SQMs structure
From the sharp breakthrough event observed at low F b values, AFM-FS for the SQMs suggests a typical behavior of a fluid membrane at RT.This type of breakthrough is characteristic of fluid state phospholipid bilayers. 42In addition, no thermal transition is detected for QS_H 2 O or QS_PBS by differential scanning calorimetry for temperatures above RT.This is consistent with previous MD simulations in which the QS components were found to diffuse with similar diffusion coefficient of typical fluid phospholipidic membranes. 33Still, to understand the origin of the coexisting domains observed in SQMs, we studied the morphology of QS_H 2 O and QS_PBS membranes by AFM at different temperatures.
When imaging the SQM in PBS/NaCl, we let the system stabilize to the homogeneous phase at RT, knowing its dynamic behavior at this T. Unsurprisingly, further increase in T up to 45 ºC showed no changes in the QS_PBS membrane topography (Figure S5).Conversely, the QS_H 2 O membrane showed a gradual transition from a heterogeneous to a homogeneous topography upon rising T from RT up to 42.5 ºC (Figure 5a and b).Increasing the T stepwise and leaving the membrane at least during 30 min at each temperature, showed that SQMs in water turned into a homogeneous phase around T=30 ºC (Figure 5c and d).Regarding this interpretation, it is worth noting that pure CTAB adsorbed onto mica can form bilayers in which the molecules have a substantial tilt of 44° to the surface normal. 50,51 he bilayer with a well-defined tilt angle is difficult to observe at RT, since this temperature coincides with the Krafft temperature of CTAB (25ºC) and near the Krafft temperature the dynamics of CTA + chains is very slow.Previous work shows that the formation of the CTA + bilayers at 25ºC has very long equilibration times (6-24 h), and other metastable structures can be observed during equilibration. 52It has been also proposed that in lipid bilayers with small Chol concentrations, where all Chol molecules interact independently with the lipid bilayer, Chol has a tilt angle of ∼10º and may influence the tilt of the other membrane components. 53In the QS_H 2 O, the bilayer is formed by CTA + and Chol that are known to interact strongly forming a 1:1 bimolecular synthon, 29 so it seems possible a complex behaviour for the tilt angle of the components, responsible for the formation of the domains with different tilt proposed in Figure 6.This interpretation is also consistent with the atomistic simulations discussed in the next section.

Molecular Dynamics (MD) Simulations
We performed all-atomic MD simulations to provide an atomistic interpretation of the experimental results, regarding both the possible penetration of the ions and the possible changes in tilt of the bilayer components.To this end, we have extended to larger simulation times our previous all-atomic MD simulations of QS_H 2 O bilayer at 25ºC. 29In these simulations, we considered a small bilayer patch (~15.7 nm 2 ) with a 1:1 mixture of CTAB surfactant and Chol in water (there is no supporting surface in the simulations).All technical details of the simulation are the same as in our previous work, 29 with the only difference that the simulation was extended for an additional time of 106 ns.In addition, we have also performed a second simulation in order to evaluate the effect of added salt.In this second simulation, we have added 100 mM of NaCl to our previous simulation of the QS_H 2 O bilayer at 25ºC (see methods for details).
The simulations of the QS_H 2 O system show that Br -counterions are not only able to adsorb on top of the bilayer but also to penetrate in the hydrophilic region of the bilayer.In the case of the simulations with added salt (100 mM of NaCl) we also observe that both anions (Br -counterions and the Cl -anions from added salt) penetrate in the hydrophilic region of the bilayer.The results for the organization of the anions in both simulations are summarized in Figure 7.
As seen in Figure 7a, the anions that penetrate inside the bilayer are shared between the CTA + headgroup and the -OH group from the Chol.In other words, anions not only interact strongly with cationic CTA + surfactant but also with Chol.It is also worth noting that previous experimental and simulation work on pure Chol monolayers has shown a strong interaction between Cl -and Chol. 49Here the simulations indicate that the addition of NaCl produces not only an incorporation of Cl -inside the bilayer but also an incorporation of Br -inside the bilayer.
In the simulations of the QS_H 2 O system (being the Br -counterions the only ions present), we observe that 10% of CTA + molecules have a Br -ion adsorbed on top and a 72% of the CTA + molecules have a Br -ion shared with cholesterol (Figure 7b).In the simulations with added NaCl, we observe that 8% of CTA + molecules have a Br -adsorbed on top and 2% of CTA + molecules have Cl -anions adsorbed on top, while a 66% Br -and 9% have Cl -ions are shared with Chol (hence a total of 75% of CTA + -Chol synthons share an anion).These simulations results are thus compatible with the experimentally observed increase in the F b after the addition of ions (Figures 2, 3 and 4), which is interpreted as due to the penetration of ions into the hydrophilic region of the QS bilayer (the one occupied by the headgroups) and affect the bilayer structure, as generally seen in phospholipid bilayers. 42,46,47,54,55  Ch the tilt was always the same than the one reported for CTA + (data not shown), so the tilt angles in Figure 8 correspond to the tilt of the CTA + -Chol synthon.The first remarkable observation is that the symmetry within the bilayer is spontaneously broken, in the sense that the CTA + surfactant molecules have different tilt in each leaflet of the bilayer.It is interesting to note that an analogous symmetry breaking has been previously observed in MD simulations of other stable vesicles (catanionic surfactant vesicles).56 More importantly, not only the average tilt angle is different in each leaflet of the bilayer but also the behavior of the tilt angle as a function of time is different.In one of the leaflets, the CTA + fluctuates around an average tilt of 10º (Figure 8).In the other leaflet, the CTA + molecules jump between states with different tilt (∼13º and 15º), remaining in these states during times of 20-30 ns, which are substantial at the scale of the simulated system.In the case of simulations with added NaCl (Figure S6 and S7), we observe again that CTA + molecules from the two leaflets have different average tilt angles (10º and 14º respectively) but the ∼20 ns jumps observed in absence of salt are not observed in presence of added salt.In this case, the fluctuation between different orientations take place at much shorter time scales, of the order of the ns or less (Figure S6).
Formation of heterogeneities (domains coexistence) is not observable by MD simulations due to the small size of the simulated systems (15.7 nm 2 ).59][60] Still, the existence of different orientations for the CTA + (and Chol) molecule in the small simulated QS_H 2 O systems suggest the possibility of the existence of different nanoscale domains with different tilt angle for the molecules in the QS_H 2 O SQMs as proposed in Figure 6 from AFM results.It is also interesting to note that according to the experimental results (Figure 2) the addition of salt suppresses the presence of heterogeneous domains.This is also consistent with the simulation results shown in Figure S6 (absence of long-lived different states with different tilt angle).

CONCLUSIONS
We characterized for the first time the topography and the nanomechanical properties of supported QS membranes in different liquid environment and T. We determined experimentally that the QS membrane behaves as a typical fluid-like phospholipid bilayer.A phase-segregated topography, stable with time, was observed by AFM in supported QS_H 2 O membranes, while in the presence of salts supported QS_PBS membranes showed a dynamic behavior from a heterogeneous topography turning into a homogeneous phase at RT.
By means of AFM-FS, we showed that the membrane breaks upon indentation with the AFM tip, as observed for 2D ordered systems like lipid bilayers and we further determined the effect of the presence of ions into the liquid media over the nanomechanical resistance of the QS membrane.
We observed by MD simulations that the anions from solution (both Br -and Cl -) not only adsorb onto the bilayer but also penetrate the hydrophilic region of the bilayer.As a result, an enhancement of the lateral interactions between the membrane molecules may be the responsible of the greater resistance to be indented by the AFM tip (higher F b ) in the presence of salts.
We demonstrate that the phase coexistence observed at RT in the QS_H .

Supported membranes
SQMs were obtained by direct fusion onto freshly cleaved mica surfaces (mica discs, Ted Pella, Redding, CA) previously glued onto Teflon discs using epoxy-based mounting glue.100 µL of QS suspension (2.75 mg ml -1 ) were deposited on to the mica for 30 min at RT. Afterwards, the samples were rinsed several times with water or PBS/NaCl buffer to remove unfused vesicles, keeping always the samples hydrated.

Atomic force microscopy (AFM) and spectroscopy (AFM-FS)
AFM images and force spectroscopy experiments were performed using an MFP-3D atomic force microscope (Asylum Research) using V-shaped Si 3 N 4 cantilevers with Si 3 N 4 tips and nominal spring constants of 0.35 N m -1 or 0.24 N m -1 (DNP, Bruker AFM Probes).The cantilever spring constants were individually calibrated using the equipartition theorem (thermal noise routine) 62 in air conditions, after measuring the sensitivity (V m -1 ) on a silicon substrate.
The same equipartition theorem was afterwards employed again to calculate the sensitivity on the required liquid environment.When required, temperature control was achieved with a Tcontrolled sample stage (BioHeater, Asylum Research).
AFM images over areas from 0.5 x 0.5 to 5 x 5 µm 2 were acquired in AC mode at RT and under liquid conditions (Milli-Q water or PBS/NaCl buffer solution).After imaging the selected region, AFM-FS was performed by approaching and retracting the AFM tip to the sample at a constant velocity of 1 µm s -1 .The force-separation curves were recorded by following an array of points from 20 x 20 to 30 x 30 (force map mode) over an imaged area.A home-made Python program based on ref. 63 was used to analyze the force-separation curves from the grids and evaluate the breakthrough force (F b ) values.Mean F b values are obtained from the gaussian fits and expressed ± SD.The membrane thickness was calculated from the force-separation curves, taking the distance between the tip-membrane initial contact and the point at which tip and mica are in contact (Figure S2).

Molecular dynamics (MD)
MD simulations consist of solving numerically the Newton equations of motion for a molecular system.In our simulations, we describe all chemical species (water, Chol, CTAB and ions) with full atomistic detail.The simulated system QS_H 2 O consists of two leaflets of 27 CTA + and 27 Chol molecules (equilibrium area ~15.7 nm 2 ) each and 54 Br -ions immersed in 5443 TIP3P water molecules.The simulations were performed using the NPTγ ensemble maintaining the QS bilayer at 25ºC, 1 atm of pressure and zero tension to mimic a vesicle bilayer.The employed force field and all technical details of the simulation are the same as in our previous work, 29 with the only difference that the simulation was extended for an additional time of 106 ns using the NAMD 2.11 software. 64 and tilt angles of CTA + with water and with added salt.This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 1 .
Figure 1.Consecutive AFM AC mode topographical images for SQM on mica in ultrapure

Figure 4 .
Figure 4. AFM AC mode topographical images (a) and AFM-FS results (F b maps (b) and

Figure 5 .
Figure 5. AFM AC-mode topographical images for QS_H 2 O membrane supported on mica, in

Figure 6 .
Figure 6.Molecular interpretation of the different topographical domains observed in supported QS_H 2 O membranes by AFM, as domains with different tilt for the CTA + -Chol bimolecular synthon.The CTA + and Chol structures are shown in CPK representation.

Figure 7 .
Figure 7. Interaction of anions with the QS components according to MD simulations at 25ºC.a) Radial correlation function g(r) computed between the anions (Br -or Cl -) and the polar headgroups (O atom of Chol or N atom of CTA + ) calculated from simulations.b) Snapshots of

Figure 8 .
Figure 8. Results obtained in MD simulations of a QS_H 2 O bilayer at 25 ºC (no added salt).The left plot shows the tilt angle for CTA + molecules (averaged over a leaflet) vs. time during the An additional simulation with added salt was performed starting from the previous simulation QS_H 2 O (with no added salt).Starting from an equilibrated configuration of the QS_H 2 O, we added 10 Na + and 10 Cl -ions (roughly corresponding to ∼100 mM) using the ionize plugin of the VMD program.After equilibration and thermalization, we ran a simulation of 131 ns employing the same parameters and conditions as in the QS_H 2 O case.The analysis of both simulations (snapshots, radial correlation functions, …) was performed using VMD software. 65ASSOCIATED CONTENT Supporting Information.QS vesicle structural characterization.Schematic figure on AFM-FS on lipid membranes.Mechanical properties of DOPC:Chol (80:20) membrane.Additional results on AFM characterization of SQMs from QS_H 2 O after changing to PBS/NaCl and SQMs from QS_PBS with T. Additional results on MD simulations of a QS bilayer with added salt (NaCl)

Table 1 .
Composition of vesicles suspensions used to prepare the SQMs.
2O SQM turns into a homogeneous structure when temperature is raised above 30ºC.All-atomic MD simulations of QS bilayer at 25ºC in absence of added salts showed that the symmetry within the bilayer is spontaneously broken, with different molecular tilt in each leaflet, one of the leaflets jumping within two possible orientations within 20-30 ns.The coexistence of different nanoscale domains may therefore be associated to different tilt angle for the molecules in the QS_H 2 O SQMs measured with AFM.The nanomechanical behavior observed indicate that QS are formed by a bilayer membrane with a compact structure homogeneous in composition, and with comparable properties to fluid-like lipid bilayers, but with the benefit of a great colloidal stability.The variations observed with the incorporation of salts suggest that the membrane properties may be easily tuned by altering the Parajo, M. F.; Ventosa, N.; Veciana, J. Multifunc-tional Nanovesicle-Bioactive Conjugates Prepared by a One-Step Scalable Method Using CO2-Expanded Solvents.Nano Lett.2013, 13, 3766−3774) and (Grimaldi, N.; Andrade, F.; Segovia, N.; Ferrer-Tasies, L.; Sala, S.; Veciana, J.; Ventosa, N. Lipid-based Nanovesicles for Nano-medicine.Chem.Soc.Rev. 2016, 45, 6520−6545.)Briefly, a 7.5 mL high-pressure vessel was loaded with a solution of 76 mg of Chol in 2.88 mL of ethanol at atmospheric pressure and 35 °C.Then, the reactor vessel was pressurized with compressed CO 2 , producing a volumetric expanded liquid solution, at a pressure of 10 MPa, a CO 2 molar fraction of X CO2 = 0.62 and a temperature of 35 °C.The system was kept at 35 °C and 10 MPa for 1 h.Finally, the CO 2 -expanded Chol solution was removed from the reactor through a depressurization valve and collected in 24 mL of aqueous solution, either U.; Vazquez, E.; Abasolo, I.; Schwartz, S.; Villaverde, A.; Albericio, F.; Royo, M.; Garcia-ultrapure water or PBS/NaCl pH 7.4 (94 mM NaCl, 4 mM PBS) buffer depending on the formulation, with 72 mg of dissolved CTAB.In this final step, where the Chol:CTAB quatsomes are formed, a flow of N 2 is used as a plunger to push down the CO 2 -expanded solution from the vessel and to maintain a constant pressure inside the vessel during depressurization.The molar ratio between the CTAB and the Chol in the final formulation was 1 to 1, which has been shown to be the correct proportion in order to have a pure vesicular phase (Ferrer-Tasies, L.; Moreno-Calvo, E.; Cano-Sarabia, M.; Aguilella-Arzo, M.; Angelova, A.; Lesieur, S.; Ricart, S.; Faraudo, J.; Ventosa, N.; Veciana, J. Quatsomes: Vesicles Formed by Self-Assembly of Sterols and Quaternary Ammonium Surfactants.Langmuir 2013, 29, 6519−6528).